U.S. patent application number 16/227690 was filed with the patent office on 2019-05-23 for ink-jet printing of tissues.
The applicant listed for this patent is WAKE FOREST UNIVERSITY HEALTH SCIENCES. Invention is credited to Anthony Atala, Tao Xu, James Yoo.
Application Number | 20190153382 16/227690 |
Document ID | / |
Family ID | 38625600 |
Filed Date | 2019-05-23 |
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United States Patent
Application |
20190153382 |
Kind Code |
A1 |
Yoo; James ; et al. |
May 23, 2019 |
INK-JET PRINTING OF TISSUES
Abstract
A method of forming an array of viable cells is carried out by
ink-jet printing a cellular composition containing said cells on a
substrate. At least two different types of viable mammalian cells
are printed on the substrate, the at least two different types of
viable mammalian cells selected to together form a tissue. In some
embodiments at least three or four different viable mammalian cells
are printed on the substrate, the cells selected to together form a
tissue. In some embodiments one of the viable mammalian cell types
is a stem cell. In some embodiments the method further comprises
printing at least one support compound on the substrate, the
support compound selected to form a tissue together with said
cells. In some embodiments the method further comprises printing at
least one growth factor on the substrate, the growth factor
selected to cause the cells to form a tissue.
Inventors: |
Yoo; James; (Winston-Salem,
NC) ; Xu; Tao; (Winston-Salem, NC) ; Atala;
Anthony; (Winston-Salem, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WAKE FOREST UNIVERSITY HEALTH SCIENCES |
Winston-Salem |
NC |
US |
|
|
Family ID: |
38625600 |
Appl. No.: |
16/227690 |
Filed: |
December 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12293490 |
Apr 23, 2009 |
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PCT/US2007/009612 |
Apr 20, 2007 |
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16227690 |
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60794033 |
Apr 21, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
B33Y 80/00 20141201; C12N 2533/54 20130101; B33Y 10/00 20141201;
B01L 3/0268 20130101; B29C 70/76 20130101; C12N 5/0062 20130101;
C12N 2533/74 20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00; B33Y 10/00 20060101 B33Y010/00; B29C 70/76 20060101
B29C070/76 |
Claims
1. In a method of forming an array of viable cells by ink-jet
printing a cellular composition containing said cells on a
substrate, the improvement comprising: printing at least two
different types of viable mammalian cells on said substrate, said
at least two different types of viable mammalian cells selected to
together form a tissue.
2. The method of claim 1, wherein at least three different viable
mammalian cells types are printed on said substrate, the cells
selected to together form a tissue.
3. The method of claim 1, wherein at least one of said viable
mammalian cell types is a stem cell.
4. The method of claim 1, further comprising printing at least one
support compound on said substrate, said support compound selected
to form a tissue together with said cells.
5. The method of claim 1, wherein said tissue is selected from the
group consisting of nerve, skin, pancreatic islet, and bone
tissue.
6. The method of claim 1, wherein said tissue is skin tissue.
7. The method of claim 1, wherein said tissue is bone tissue.
8. The method of claim 1, wherein said tissue is pancreatic islet
tissue.
9. The method of claim 1, wherein said tissue is nerve tissue.
10. In a method of forming an array of viable cells by ink-jet
printing a cellular composition containing said cells on a
substrate, the improvement comprising: printing viable stem cells
on said substrate.
11. The method of claim 10, wherein said stem cells are amniotic
fluid stem cells.
12. The method of claim 1, further comprising the step of
implanting said array in vivo in a subject in need thereof.
13. The method of claim 12, further comprising maintaining said
array in vivo in said subject for at least one month, during which
all cell types in said array maintain their structural and spatial
orientation in vivo.
14. The method of claim 12, further comprising maintaining said
array in vivo in said subject for at least two months, during which
all cell types in said array maintain their structural and spatial
orientation in vivo and retain their cellular characteristics and
tissue function.
15. In a method of forming an array of viable cells by ink jet
printing a cellular composition containing said cells on a
substrate, the improvement comprising: printing viable cancer cells
on said substrate.
16. The method of claim 15, wherein said cancer cells are selected
from the group consisting of leukemia, lymphoma, breast, lung,
colon, prostate, ovarian, skin, melanoma, and brain cancer cells.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns ink-jet printing of viable
cells and arrays of cells so produced.
BACKGROUND OF THE INVENTION
[0002] Living tissues maintain an inherent multi-cellular
heterogeneous structure. Rebuilding of such complex structure
requires subtle arrangements of different types of cells and
extracellular matrices (ECM) at their specific anatomical target
sites. To achieve tissue reconstitution, an effective method for a
precise delivery of cells and biomaterials is needed. The inkjet
printing technology has been applied to address this endeavor.
[0003] The following references are noted herein:
[0004] T. Boland et al., Ink jet printing of viable cells, U.S.
Pat. No. 7,051,654;
[0005] W. Warren et al., Architecture tool and methods of use, U.S.
Pat. No. 6,986,739; and
[0006] J. Barron et al., Biological laser printing via indirect
photon-biomaterial interactions, US Patent Application Publication
No. 2005/0018036.
SUMMARY OF THE INVENTION
[0007] Although the capability of inkjet printing of viable single
cells has been verified, the possibility of simultaneously printing
multiple cell types to build viable heterogeneous cellular
constructs has not been demonstrated to date. It has been found
that distinct cell types can be mixed with support compounds
(collagen gels) and printed into the target areas to form
3-dimensional tissue structures. Further, basic physiological
functions and properties of each cell type within the structure can
be maintained.
[0008] A first aspect of the invention is, in a method of forming
an array of viable cells by ink-jet printing a cellular composition
containing said cells on a substrate, the improvement comprising
printing at least two different types of viable mammalian cells on
said substrate, said at least two different types of viable
mammalian cells selected to together form a tissue. In some
embodiments at least three or four different viable mammalian cells
are printed on said substrate, the cells selected to together form
a tissue. In some embodiments one of said viable mammalian cell
types is a stem cell. In some embodiments the method further
comprises printing at least one support compound on said substrate,
said support compound selected to form a tissue together with said
cells. In some embodiments the method further comprises printing at
least one growth factor on said substrate, the growth factor
selected to cause the cells to form a tissue.
[0009] Example tissues, or tissue substitutes, that may be produced
by the processes of the invention include nerve, skin, pancreatic
islet, and bone tissue.
[0010] Since it is preferred to print three-dimensional arrays when
forming tissues or tissue substitutes as described above, and since
such printing may require substantially greater times than required
in prior techniques, it is sometimes preferred to carry out the
printing in a culture chamber or an environmentally controllable
chamber to enhance the survival of cells after printing.
[0011] Another aspect of the invention is a method of forming an
array of viable cells by ink-jet printing a cellular composition
containing said cells on a substrate, the improvement comprising:
printing viable stem cells (for example, amniotic fluid stem cells)
on the substrate.
[0012] Another aspect of the invention is a method of forming an
array of viable cells by ink jet printing a cellular composition
containing said cells on a substrate, the improvement comprising:
printing viable cancer cells on said substrate. Arrays produced by
such methods are useful in screening compounds for efficacy in
treating cancer by contacting the compound to the cancer cells.
Arrays can be printed with normal or "control" cells adjacent to
the cancer cells, so that the test compound may be concurrently
contacted to the control cells, so that the differential effect of
the test compound on cancer cells as compared to control cells may
be evaluated.
[0013] The foregoing and other objects and aspects of the present
invention are explained in greater detail in the drawings herein
and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. (a) Multi-cellular "pie" structure. Morphologies of
3 distinct cell types in the "pie" structure: (b) SMC, (c) hAFSC,
and (d) MS1. (e) Printed collagen scaffold with different color
dyes.
[0015] FIG. 2. (a) Alizarin red staining of printed hAFSC within
the 3-D collagen constructs after 25 days of culture. (b) I-V
relationship of BSMC printed in the collagen constructs.
[0016] FIG. 3. (a) Fluorescent microscopic image of a 3-D "pie"
before implantation. (b) Gross view of the retrieved "pie" 2 weeks
post-implantation. (c) and (d) Fluorescent images of ECs and SMCs
within the "pie" implant, respectively.
[0017] FIG. 4. (a) I-V relationship of printed SMCs before and 4
weeks after implantation. (b) MRI scanning of EC-printed constructs
8 weeks after implantation. (c) Micro-CT scanning of AFSC-printed
samples 18 weeks after implantation. New engineered bone tissues
were observed within the implants. (d) and (e) Immunohistochemical
analyses of EC-printed constructs 8 weeks after implantation and
AFSC-printed constructs 18 week after implantation,
respectively.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] "Support compound" as used herein may be any naturally
occurring or synthetic support compound, including combinations
thereof, suitable for the particular tissue or array being printed.
In general the support compound is preferably physiologically
acceptable or biocompatible. Suitable examples include but are not
limited to alginate, collagen (including collagen VI), elastin,
keratin, fibronectin, proteoglycans, glycoproteins, polylactide,
polyethylene glycol, polycaprolactone, polycolide, polydioxanone,
polyacrylates, polysulfones, peptide sequences, proteins and
derivatives, oligopeptides, gelatin, elastin, fibrin, laminin,
polymethacrylates, polyacetates, polyesters, polyamides,
polycarbonates, polyanhydrides, polyamino acids carbohydrates,
polysaccharides and modified polysaccharides, and derivatives and
copolymers thereof (see, e.g., U.S. Pat. Nos. 6,991,652 and
6,969,480) as well as inorganic materials such as glass such as
bioactive glass, ceramic, silica, alumina, calcite, hydroxyapatite,
calcium phosphate, bone, and combinations of all of the
foregoing.
[0019] "Growth factor" as used herein may be any naturally
occurring or synthetic growth factor, including combinations
thereof, suitable for the particular tissue or array being printed.
Numerous growth factors are known to those skilled in the art.
Examples include but are not limited to: insulin-like growth factor
(e.g., IGF-1), transforming growth factor-beta (TGF-beta),
bone-morphogenetic protein, fibroblast growth factor, platelet
derived growth factor (PDGF), vascular-endothelial growth factor
(VEGF), connective tissue growth factor (CTGF), basic fibroblast
growth factor (bFGF), epidermal growth factor, fibroblast growth
factor (FGF) (numbers 1, 2 and 3), osteopontin, bone morphogenetic
protein-2, growth hormones such as somatotropin, cellular
attractants and attachment agents, etc., and mixtures thereof. See,
e.g., U.S. Pat. Nos. 7,019,192; 6,995,013; and 6,923,833.
[0020] "Cells" as used herein may be of any suitable species, and
in some embodiments are of the same species as the subject into
which tissues produced by the processes herein are implanted.
Mammalian cells (including mouse, rat, dog, cat, monkey and human
cells) are in some embodiments particularly preferred.
[0021] "Stem cell" as used herein refers to a cell that has the
ability to replicate through numerous population doublings (e.g.,
at least 60-80), in some cases essentially indefinitely, and to
differentiate into multiple cell types (e.g., is pluripotent or
multipotent).
[0022] "Embryonic stem cell" as used herein refers to a cell that
is derived from the inner cell mass of a blastocyst and that is
pluripotent.
[0023] "Amniotic fluid stem cell" as used herein refers to a cell,
or progeny of a cell, that (a) is found in, or is collected from,
mammalian amniotic fluid, mammalian chorionic villus, and/or
mammalian placental tissue, or any other suitable tissue or fluid
from a mammalian donor, (b) is pluripotent; (c) has substantial
proliferative potential, (d) optionally, but preferably, does not
require feeder cell layers to grow in vitro, (e) optionally, but
preferably, specifically binds c-kit antibodies (particularly at
the time of collection, as the ability of the cells to bind c-kit
antibodies may be lost over time as the cells are grown in
vitro).
[0024] "Pluripotent" as used herein refers to a cell that has
complete differentiation versatility, e.g., the capacity to grow
into any of the animals cell types. A pluripotent cell can be
self-renewing, and can remain dormant or quiescent with a tissue.
Unlike a totipotent cell (e.g., a fertilized, diploid egg cell) a
pluripotent cell cannot usually form a new blastocyst.
[0025] "Multipotent" as used herein refers to a cell that has the
capacity to grow into any of a subset of the corresponding animals
cell type. Unlike a pluripotent cell, a multipotent cell does not
have the capacity to form all of the cell types of the
corresponding animal.
[0026] "Cancer cells" as used herein may be of any type, including
but not limited to leukemia, lymphoma, breast, lung, colon,
prostate, ovarian, skin, melanoma, and brain cancer cells.
[0027] Subjects that may be implanted with constructs or arrays of
the present invention include both human subjects and animal
subjects (particularly mammalian subjects such as dogs, cats,
horses, pigs, sheep, cows, etc.) for veterinary purposes.
[0028] The disclosures of all United States patent references cited
herein are to be incorporated herein by reference in their
entirety.
A. Tissue Printing.
[0029] Methods and compositions for the ink-jet printing of viable
cells are known and described in, for example, T. Boland et al., US
Patent Application No. 2004/0237822 (Dec. 2, 2004) (Clemson
University) and W. Wilson and T. Boland, The Anatomical Record Part
A, 272A: 491-496 (2003). While the present invention is primarily
concerned with ink-jet printing of cells, the cells may be printed
by other means as well, such as the methods and compositions for
forming three-dimensional structures by deposition of viable cells
described in W. Warren et al., U.S. Pat. No. 6,986,739 (Sciperio
Inc.).
[0030] Although not required, cells can typically be printed in the
form of a "cell composition" that contains a liquid carrier for the
cells. The cell composition can be in the form of a suspension,
solution, or any suitable form. Examples of suitable liquid
carriers include, but are not limited to, water, ionic buffer
solutions (e.g., phosphate buffer solution, citrate buffer
solution, etc.), liquid media (e.g., modified Eagle's medium
("MEM"), Hanks' Balanced Salts, etc.), and so forth. For instance,
the use of a liquid carrier in the cell composition can ensure
adequate hydration and minimize evaporation of the cells after
printing. However, the probability of obtaining viable cells in any
given printed drop also decreases with decreasing cell
concentration. (T. Boland, US Patent Application Publication No.
20040237822 at para 48)
[0031] Various mechanisms may be employed to facilitate the
survival of the cells during and/or after printing. Specifically,
compounds may be utilized that "support" the printed cells by
providing hydration, nutrients, and/or structural support. These
compounds may be applied to the substrate using conventional
techniques, such as manually, in a wash or bath, through vapor
deposition (e.g., physical or chemical vapor deposition), etc.
These compounds may also be combined with the cell composition
before and/or during printing, or may be printed or otherwise
applied to the substrate (e.g., coated) as a separate layer
beneath, above, and/or between cell layers. For example, one such
support compound is a gel having a viscosity that is low enough
under the printing conditions to pass through the nozzle of the
printer head, and that can gel to a stable shape during and/or
after printing. Such viscosities are typically within the range of
from about 0.5 to about 50 centipoise, in some embodiments from
about 1 to about 20 centipoise, and in some embodiments, from about
1 to about 10 centipoise. Some examples of suitable gels that may
be used in the present invention include, but are not limited to,
agars, collagen, hydrogels, etc. One example of a collagen gel for
facilitating cell growth is described in Collagen As a Substrate
for Cell Growth and Differentiation, Methods in Enzymology, Strom
and Michalopoulous, Vol. 82. 544-555 (1982) (T. Boland at para
50).
[0032] Besides gels, other support compounds may also be utilized
in the present invention. Extracellular matrix analogs, for
example, may be combined with support gels to optimize or
functionalize the gel. One or more growth factors may also be
introduced in the printed cell arrays. For example, slow release
microspheres that contain one or more growth factors in various
concentrations and sequences may be combined with the cell
composition to accelerate and direct the cell fusion process. Other
suitable support compounds might include those that aid in avoiding
apoptosis and necrosis of the developing structures. For example,
survival factors (e.g., basic fibroblast growth factor) may be
added. In addition, transient genetic modifications of cells having
antiapoptotic (e.g., bcl-2 and telomerase) and/or blocking pathways
may be included in cell aggregates to be printed according to the
invention. Adhesives may also be utilized to assist in the survival
of the cells after printing. For instance, soft tissue adhesives,
such a cyanoacrylate esters, fibrin sealant, and/or
gelatin-resorcinol-formaldehyde glues, may be utilized to inhibit
nascent constructs from being washed off or moved following
printing of a layer. In addition, adhesives, such as
arginine-glycine-aspartic acid ligands, may enhance the adhesion of
cells to a gelling polymer or other support compound. In addition,
extracellular proteins, extracellular protein analogs, etc., may
also be utilized (T. Boland at para 55).
[0033] Besides two-dimensional arrays, three-dimensional arrays may
also be formed. Three-dimensional cell arrays are commonly used in
tissue engineering and biotechnology for in-vitro and in-vivo cell
culturing. In general, a three-dimensional array is one which
includes two or more layers separately applied to a substrate, with
subsequent layers applied to the top surface of previous layers.
The layers can, in one embodiment, fuse or otherwise combine
following application or, alternatively, remain substantially
separate and divided following application to the substrate.
Three-dimensional arrays may be formed in a variety of ways in
accordance with the present invention. For example, in one
embodiment, three-dimensional arrays may be formed by printing
multiple layers onto the substrate. (T. Boland at para 60).
[0034] The thickness of a printed layer (e.g., cell layer, support
layer, etc.) may generally vary depending on the desired
application. For example, in some embodiments, the thickness of a
layer containing cells is from about 2 micrometers to about 3
millimeters, and in some embodiments, from about 20 micrometers to
about 100 micrometers. Further, as indicated above, support
compounds, such as gels, are often used to facilitate the survival
of printed cells. The present inventors have discovered that the
development of a cellular assembly may be increased when the
thickness of the support layer(s) (e.g., between cells) is
approximately the same as the size of the cells deposited adjacent
to the support compound (T. Boland at para 61). When printing
certain types of two-dimensional or three-dimensional arrays, it is
sometimes desired that any subsequent cell growth is substantially
limited to a predefined region. Thus, to inhibit cell growth
outside of this predefined region, compounds may be printed or
otherwise applied to the substrate that inhibit cell growth and
thus form a boundary for the printed pattern. Some examples of
suitable compounds for this purpose include, but are not limited
to, agarose, poly(isopropyl N-polyacrylamide) gels, and so forth.
In one embodiment, for instance, this "boundary technique" may be
employed to form a multi-layered, three-dimensional tube of cells,
such as blood vessels. For example, a cell suspension may be mixed
with a first gel ("Gel A") in one nozzle, while a second gel ("Gel
B") is loaded into another nozzle. Gel A induces cell attachment
and growth, while Gel B inhibits cell growth. To form a tube, Gel A
and the cell suspension are printed in a circular pattern with a
diameter and width corresponding to the diameter and wall thickness
of the tube, e.g., from about 3 to about 10 millimeters in diameter
and from about 0.5 to about 3 millimeters in wall thickness. The
inner and outer patterns are lined by Gel B defining the borders of
the cell growth. For example, a syringe containing Gel A and "CHO"
cells and a syringe containing Gel B may be connected to the
nozzle. Gel B is printed first and allowed to cool for about 1 to 5
minutes. Gel A and CHO cells are then printed on the agarose
substrate. This process may be repeated for each layer. (T. Boland
at para 62).
[0035] The present invention particularly provides for the printing
of tissues by the appropriate combination of cell and support
material, or two or three or more different cell types typically
found in a common tissue, preferably along with appropriate support
compound or compounds, and optionally but preferably with one or
more appropriate growth factors. Cells, support compounds, and
growth factors may be printed from separate nozzles or through the
same nozzle in a common composition, depending upon the particular
tissue (or tissue substitute) being formed. Printing may be
simultaneous, sequential, or any combination thereof. Some of the
ingredients may be printed in the form of a first pattern (e.g., an
erodable or degredable support material), and some of the
ingredients may be printed in the form of a second pattern (e.g.,
cells in a pattern different from the support, or two different
cell types in a different pattern). Again the particular
combination and manner of printing will depend upon the particular
tissue. Materials to be printed for specific tissues or tissue
substitutes are described further below.
[0036] Skin. In representative embodiments, to produce
epidermal-like skin tissue, the following are printed: [0037] (a)
at least one cell type, and preferably at least two or in some
embodiments three or four different epidermal cell types (e.g.,
keratinocytes, melanocytes, Merkel cells, Langerhan cells, etc.,
and any combination thereof); and/or [0038] (b) at least one
support compound such as described above (e.g., collagen, elastin,
keratin, etc., and any combination thereof); and/or [0039] (c) at
least one growth factor as described above (e.g., basic fibroblast
growth factor (bFGF), Insulin-Like Growth Factor 1, epidermal
growth factor (EGF), etc., and any combination thereof);
[0040] In some embodiments the epidermal cells, support compound
and/or growth factors printed as described above (which form an
"epidermal" type layer) are printed on, or on top of, a previously
formed (e.g., printed or ink-jet printed) "dermal" type layer, the
previously printed dermal layer layers comprising: (a) one, two,
three or four different dermal cells (fibroblasts, adipocytes, mast
cells, and/or macrophages), (b) at least one support compound as
described above; and/or (c) at least one growth factor as described
above.
[0041] Skin tissue produced by the process of the present invention
is useful for implantation into or on a subject to, for example,
treat burns, and other wounds such as incisions, lacerations, and
crush injuries (e.g., postsurgical wounds, and posttraumatic
wounds, venous leg ulcers, diabetic foot ulcers, etc.)
[0042] Bone. In particular embodiments, to produce bone tissues,
the following are printed: [0043] (a) at least one bone cell type,
and preferably at least two or three different bone cell types
(e.g., osteoblasts, osteoclasts, osteocytes, and any combination
thereof, but in some embodiments at least osteoblasts and
osteoclasts, and in some embodiments all three); and/or [0044] (b)
at least one support compound such as described above (e.g.,
collagen, hydroxyapatites, calicite, silica, ceramic,
proteoglycans, glycoproteins, etc., and any combination thereof);
and/or [0045] (c) at least one growth factor (e.g., bone
morphogenetic protein, transforming growth factor, fibroblast
growth factors, platelet-derived growth factors, insulin-like
growth factors, etc., and any combination thereof). Bone tissues
produced by the processes described herein are useful for, among
other things, implantation into a subject to treat bone fractures
or defects, and/or promote bone healing.
[0046] Pancreatic. In representative embodiments, to produce
pancreatic islet tissues, the following are printed: [0047] (a) at
least one, two, or three different pancreatic islet cell type
(e.g., glucagon-synthesizing A (.alpha.) cells, insulin-producing B
(.beta.) cells, D (.delta.) cells, etc., and any combination
thereof); and/or [0048] (b) at least one support compound such as
described above (e.g., collagen, proteoglycans, glycoproteins,
elastin, etc., and any combination thereof); and/or [0049] (c) at
least one growth factor (e.g., insulin-Like Growth Factor II
(IGF-II), gastrin, transforming growth factor-alpha (TGF alpha),
vascular endothelial growth factor (VEGF), etc., and any
combination thereof)
[0050] Pancreatic islet tissue produced by the processes described
herein is useful for, among other things, implantation into a
subject to treat diabetes (including type I and type II
diabetes).
[0051] Nerve. In representative embodiments, to produce nerve
tissue, the following are printed: [0052] (a) at least one, two or
three cells types, and preferably (i) a central or peripheral nerve
cells (e.g., cortical neurons, hippocampal neurons, dopaminergic
neurons, cholinergic neurons, adrenergic neurons, noradrenergic
neurons, etc., including any combination thererof), and/or (ii) at
least one glial cell type (e.g., neuroglia, astrocytes,
oligodendrocytes, Schwann cells, etc., including any combination
thereof) and (iii) any combination thereof (e.g., a combination of
at least one nerve cell and at least one glial cell); and/or [0053]
(b) at least one support compound such as described above; (e.g.,
laminin, collagen type IV, fibronectin, etc., and any combination
thereof); and/or [0054] (c) at least one growth factor (e.g., NGF,
brain-derived neurotrophic factor, insulin-like growth factor-I,
fibroblast growth factor, etc., or any combination thereof); and
any combination of the foregoing.
[0055] Nerve tissue produced by the processes described herein is
useful, among other things, for implantation into a subject to
treat nerve injury or degenrative diseases such as Parkinson's
disease and Alzheimer's disease.
B. Stem cells.
[0056] In some embodiments stem cells are printed onto substrates
by ink-jet printing. Stem cells may be printed alone (typically in
combination with a support compound or compounds) or in combination
with one or more additional cells (e.g., in a combination selected
to produce a tissue as described above).
[0057] Stem cells (such as pluripotent or multipotent cells) are
capable of differentiating into multiple different cell types or
lines, including at least one of a hepatogenic-specific (or
liver-specific) cell line, a myogenic (or muscle specific) cell
line, an osteogenic (or bone specific) cell line, or an endothelial
specific cell line. Useful cells for carrying out the invention
include but are not limited to embryonic stem cells,
parthenogenetic stem cells, amniotic fluid stem cells, and
adipose-derived stem cells.
[0058] Embryonic stem cells useful for carrying out the present
invention are known and described in, for example, U.S. Pat. No.
6,200,806 to Thomson and U.S. Pat. No. 5,843,780 to Thomson.
[0059] Adipose-derived stem cells are known and described in, for
example, U.S. Pat. No. 6,777,231 to Katz et al.
[0060] Parthenogenetic stem cells useful for carrying out the
present invention are known and described in, for example, J. Hipp
et al., Parthenogenetic Stem Cells, in Myers, R. A. (Ed.): Meyers
Encyclopedia of Molecular Cell Biology and Molecular Medicine, Vol.
10, pp. 71-84 (2d Ed. 2005) and K. Vrana et al., Non-human Primate
Parthenogenetic Stem Cells, Proc. Natl. Acad. Sci. USA 100 Suppl 1:
11911-6 (2003).
[0061] Amniotic fluid stem cells (AFSCs) useful for carrying out
the present invention are known and described in, for example, PCT
Application WO 03/042405 to Atala and DeCoppi; In 't Anker, P. S.,
et al., Amniotic fluid as a novel source of mesenchymal stem cells
for therapeutic transplantation. Blood, 2003. 102(4): p. 1548-9;
Prusa, A. R., et al., Oct-4-expressing cells in human amniotic
fluid: a new source for stem cell research? Hum Reprod, 2003.
18(7): p. 1489-93; Kaviani, A., et al., The amniotic fluid as a
source of cells for fetal tissue engineering. J Pediatr Surg, 2001.
36(11): p. 1662-5; Prusa, A. R. and M. Hengstschlager, Amniotic
fluid cells and human stem cell research: a new connection. Med Sci
Monit, 2002. 8(11): p. RA253-7.
[0062] In general, AFSCs are cells, or progeny of cells, that are
found in or collected primarily from mammalian amniotic fluid, but
may also be collected from mammalian chorionic villus or mammalian
placental tissue. The cells are preferably collected during the
second trimester of gestation. In mice the cells are most
preferably collected during days 11 and 12 of gestation. Preferably
the mammalian source is of the same species as the mammalian
subject being treated.
[0063] In general, the tissue or fluid can be withdrawn by
amniocentesis, punch-biopsy, homogenizing the placenta or a portion
thereof, or other tissue sampling techniques, in accordance with
known techniques. From the sample, stem cells or pluripotent cells
may be isolated with the use of a particular marker or selection
antibody that specifically binds stem cells, in accordance with
known techniques such as affinity binding and/or cell sorting.
Particularly suitable is the c-Kit antibody, which specifically
binds to the c-kit receptor protein. C-kit antibodies are known
(see, e.g., U.S. Pat. Nos. 6,403,559, 6,001,803, and 5,545,533).
Particularly preferred is the antibody c-Kit(E-1), a mouse
monoclonal IgG that recognizes an epitope corresponding to amino
acids 23-322 mapping near the human c-kit N-terminus, available
from Santa Cruz Biotechnology, Inc., 2145 Delaware Avenue, Santa
Cruz, Calif., USA 95060, under catalog number SC-17806).
[0064] AFSCs used to carry out the present invention are
pluripotent. Hence, they differentiate, upon appropriate
stimulation, into at least osteogenic, adipogenic, myogenic,
neurogenic, hematopoitic, and endothelial cells. Appropriate
stimulation, for example, may be as follows: Osteogenic induction:
The cKit.sup.+ cells are cultured in DMEM low glucose with 10% FBS
supplementing with 100 nM dexamethasone (Sigma-Aldrich), 10 mM
beta-glycerophosphate (Sigma-Aldrich) and 0.05 mM ascorbic
acid-2-phosphate (Wako Chemicals, Irving, Tex.); Adipogenic
induction: To promote adipogenic differentiation, c-Kit.sup.+ cells
are seeded at density of 3000 cells/cm.sup.2 in DMEN low glucose
medium with 10% FBS supplemented with 1 .mu.M dexamethasone, 1 mM
3-isobutyl-1-methylxantine, 10 .mu.g/ml insulin and 60 .mu.M
indomethacin (all from Sigma-Aldrich); Myogenic induction:
c-Kit.sup.+ cells were plated into Matrigel-precoated dish (1
mg/ml, Collaborative Biomedical Products) and cultured in myogenic
medium (DMEM low glucose supplemented with 10% horse serum, and
0.5% chick embryo extract from Gibco) followed by treatment of
5-azacytidine (10 .mu.M, Sigma) added in myogenic medium for 24 h;
Endothelial induction: c-Kit.sup.+ cells are plated into
gelatin-precoated dish and cultured in endothelial basal medium-2
(EBM-2, Clonetics BioWittaker) supplemented with 10% FBS and 1%
glutamine (Gibco). In preferred embodiments no feeder layer or
leukaemia inhibitory factor (LIF) are required either for expansion
or maintenance of AFSCs in the entire culture process.
[0065] AFSCs also have substantial proliferative potential. For
example, they proliferate through at least 60 or 80 population
doublings or more when grown in vitro. In preferred embodiments
AFSCs used to carry out the invention proliferate through 100, 200
or 300 population doublings or more when grown in vitro. In vitro
growth conditions for such determinations may be: (a) placing of
the amniotic fluid or other crude cell-containing fraction from the
mammalian source onto a 24 well Petri dish containing a culture
medium [.alpha.-MEM (Gibco) containing 15% ES-FBS, 1% glutamine and
1% Pen/Strept from Gibco supplemented with 18% Chang B and 2% Chang
C from Irvine Scientific], upon which the cells are grown to
confluence, (b) dissociating the cells by 0.05% trypsin/EDTA
(Gibco), (c) isolating an AFSC subpopulation based on expression of
a cell marker c-Kit using mini-MACS (Mitenyl Biotec Inc.), (d)
plating of cells onto a Petri dish at a density of
3-8.times.10.sup.3/cm.sup.2, and (e) maintaining the cells in
culture medium for more than the desired time or number of
population doublings.
[0066] Preferably, the AFSCs are also characterized by the ability
to be grown in vitro without the need for feeder cells (as
described in PCT Application WO 03/042405 to Atala and DeCoppi. In
preferred embodiments undifferentiated AFSCs stop proliferating
when grown to confluence in vivo.
[0067] AFSCs used to carry out the present invention are preferably
positive for alkaline phosphatase, preferably positive for Thy-1,
and preferably positive for Oct4, all of which are known markers
for embryonic stem cells, and all of which can be detected in
accordance with known techniques. See, e.g., Rossant, J., Stem
cells from the Mammalian blastocyst. Stem Cells, 2001. 19(6): p.
477-82; Prusa, A. R., et al., Oct-4-expressing cells in human
amniotic fluid: a new source for stem cell research? Hum Reprod,
2003. 18(7): p. 1489-93.
[0068] In a particularly preferred embodiment, the AFSCs do not
form a teratoma when undiferentiated AFSCs are grown in vivo. For
example, undifferentiated AFSCs do not form a teratoma within one
or two months after intraarterial injection into a 6-8 week old
mouse at a dose of 5.times.10.sup.6 cells per mouse.
[0069] In preferred embodiments the amniotic fluid stem cells used
to carry out the present invention express several markers
characteristic of ES cells and/or various multipotent adult stem
cells. These include the transcription factor Oct-4 (Pou5f1),
SSEA-1 (Stage Specific Embryonic Antigen 1), Sca-1 (Ly-6A/E), CD90
(Thy-1), and CD44 (Hyaluronate receptor. Ly-24, P-1).
[0070] In preferred embodiments the amniotic fluid stem cells used
to carry out the present invention do not express CD34 and CD105,
markers of certain lineage restricted progenitors, nor the
hematopoietic marker CD45.
[0071] In preferred embodiments the amniotic fluid stem cells used
to carry out the present invention express low levels of major
histocompatibility (MHC) Class I antigens and are negative for MHC
Class II.
[0072] Differentiation of cells. "Differentiation" and
"differentiating" as used herein include (a) treatment of the cells
to induce differentiation and completion of differentiation of the
cells in response to such treatment, both prior to printing on a
substrate, (b) treatment of the cells to induce differentiation,
then printing of the cells on a substrate, and then differentiation
of the cells in response to such treatment after they have been
printed, (c) printing of the cells, simultaneously or sequentially,
with a differentiation factor(s) that induces differentiation after
the cells have been printed, (d) contacting the cells after
printing to differentiation factors or media, etc., and
combinations of all of the foregoing. In some embodiments
differentiation may be modulated or delayed by contacting an
appropriate factor or factors to the cell in like manner as
described above. In some embodiments appropriate differentiation
factors are one or more of the growth factors described above.
Differentiation and modulation of differentiation can be carried
out in accordance with known techniques, as described in greater
detail below, or as described in U.S. Pat. No. 6,589,728, or US
Patent Application Publication Nos.: 2006006018 (endogenous repair
factor production promoters); 20060013804 (modulation of stein cell
differentiation by modulation of caspase-3 activity); 20050266553
(methods of regulating differentiation in stem cells); 20050227353
(methods of inducing differentiation of stem cells); 20050202428
(pluripotent stem cells); 20050153941 (cell differentiation
inhibiting agent, cell culture method using the same, culture
medium, and cultured cell line); 20050131212 (neural regeneration
peptides and methods for their use in treatment of brain damage);
20040241856 (methods and compositions for modulating stem cells);
20040214319 (methods of regulating differentiation in stem cells);
20040161412 (cell-based VEGF delivery); 20040115810 (stem cell
differentiation-inducing promoter); 20040053869 (stem cell
differentiation); or variations of the above or below that will be
apparent to those skilled in the art.
[0073] Pancreas. Differentiation of cells to pancreatic-like cells
can be carried out in accordance with any of a variety of known
techniques. For example, the cells can be contacted to, printed
with, or cultured in a conditioning media such as described in US
Patent Application 2002/0182728 (e.g., a medium that comprises
Dulbecco's Minimal Essential Medium (DMEM) with high glucose and
sodium pyruvate, bovine serum albumin, 2-mercaptoethanol, fetal
calf serum (FCS), penicillin and streptomycin (Pen-Strep), and
insulin, transferrin and selenium). In another example, the cells
may be treated with a cAMP upregulating agent to induce
differentiation as described in U.S. Pat. No. 6,610,535 to Lu. In
still another example, the cells may be grown in a reprogramming
media, such as described in US Patent Application 2003/0046722A1 to
Collas to induce differentiation to a pancreatic cell type. In
another embodiment, differentiation may be carried out using the 5
steps protocol describe by Lumelsky at al. Lumelsky, N., et al.,
Differentiation of embryonic stem cells to insulin-secreting
structures similar to pancreatic islets. Science, 2001. 292(5520):
p. 1389-94. In another embodiment, differentiation may be carried
out using DMSO to induce pancreatic differentiation in vitro.
She-Hoon Oh et al, Adult bone marrow-derived cells
trans-differentiating into insulin producing cells for the
treatment of type I diabetes. Lab Inv, 2004, 84: 607-617. In
another embodiment, differentiation may be carried out using
Nicotinamide to induce pancreatic differentiation in vitro. See,
e.g., Otonkoski, T., et al, Nicotinamide is a potent inducer of
endocrine differentiation in cultured human fetal pancreatic cells.
J Clin Invest, 1993, 92(3): 1459-1466. In another embodiment,
differentiation may be carried out using inhibitors of
phosphoinositide 3-kinase (PI3K), such as LY294002, to induce
pancreatic differentiation in vitro. See, e.g., Hori, Y., et al.,
Growth inhibitor promote differentiation of insulin producing
tissue from embryonic stem cells. PNAS, 2002, 99:16105-16110. In
another embodiment Exendin-4, a naturally occurring 39-amino acid
peptide originally isolated from the salivary secretions of the
Lizard Heloderma suspectum, can be used to induce pancreatic
differentiation in vitro. Nielsen, L L., et al., Pharmacology of
exenatide (synthetic exendin-4): a potential therapeutic for
improved glycemic control of type 2 diabetes. Regul Pept. 2004 Feb
15;117(2):77-88. Review. In still another embodiment, anti-sonic
hedgehog (Anti-Shh) and co-culturing with pancreatic rudiments can
be used to induce pancreatic differentiation in vitro. Leon-Quinto,
T., et al., In vitro direct differentiation of mouse embryonic stem
cells into insulin producing cells. Diabetologia, 2004,
47:1442-1451. In one preferred embodiment the differenting step is
carried out by transducing (sometimes also referred to as
"engineering" or "transforming") the cells with a vector, or
introducing into the cells a vector, that contains a nucleic acid
encoding a differentiation factor (such as Pdx1, Ngn3, Nkx6.1,
Nkx2.1, Pax6, or Pax4) and expresses the differentiation factor in
the cells, or by activating the expression of an endogeneous
nucleic acid encoding a differentiation factor in the cells (e.g.,
engineering the cells to activate transcription of an endogeneous
differentiation factor such as Pdx1, Ngn3, Nkx6.1, Nkx2.1, Pax6, or
Pax4, such as by inserting a heterologous promoter in operative
associated with an endogeneous differentiation factor, in
accordance with known techniques. See, e.g., U.S. Pat. No.
5,618,698). Such exogeneous nucleic acids may be of any suitable
source, typically mammalian, including but not limited to rodent
(mouse, hamster, rat), dog, cat, primate (human, monkey), etc.
[0074] Osteogenic induction: Cells may be induced to form bone
cells by any suitable technique, such as culturing in DMEN low
glucose with 10% FBS supplementing with 100 nM dexamethasone
(Sigma-Aldrich), 10 mM beta-glycerophosphate (Sigma-Aldrich) and
0.05 mM ascorbic acid-2-phosphate (Wako Chemicals, Irving,
Tex.).
[0075] Adipogenic induction: Cells may be induced to promote
adipogenic differentiation by any suitable technique, such as
culturing in DMEN low glucose medium with 10% FBS supplemented with
1 .mu.M dexamethasone, 1 mM 3-isobutyl-1-methylxantine, 10 .mu.g/ml
insulin and 60 .mu.M indomethacin (all from Sigma-Aldrich);
[0076] Myogenic induction: Cells may be induced to promote myogenic
induction by any suitable technique, such as culturing in myogenic
medium (DMEM low glucose supplemented with 10% horse serum, and
0.5% chick embryo extract from Gibco) followed by treatment of
5-azacytidine (10 .mu.M, Sigma) added in myogenic medium for 24
h.
[0077] Endothelial induction: Cells may be induced to promote
endothelial induction by any suitable technique, such as culturing
in endothelial basal medium-2 (EBM-2, Clonetics BioWittaker)
supplemented with 10% FBS and 1% glutamine (Gibco).
[0078] The present invention is explained in greater detail in the
following non-limiting examples.
EXAMPLE 1
Printing of Multiple Cell Types
[0079] Materials and Methods. Three distinct cell types were used
in this study: human amniotic fluid-derived stem cells (hAFSC)
transfected with lacZ, bladder smooth muscle cells (BSMC), and GFP
labeled MS1 (mouse pancreatic islet endothelial cell line). Each
cell type was grown separately, trypsinized, collected and
resuspended in Type I collagen solution. Different mixtures of
collagen and cells were loaded into different ink cartridges. Each
cell-collagen mixture was printed layer-by-layer into the
pre-designed target locations using a modified HP 550 printer. A
solution containing NaOH was subsequently printed in order to
neutralize the pH. The printed constructs were placed in the
incubator for 3-5 hours. Once the collagen gel was set, 3-D viable
multi-cellular constructs with a specific shape were formed. After
2 days of culture, the printed multi-cellular constructs were fixed
and characterized using cell specific markers (.alpha.-actin,
X-gal).
[0080] To examine the function of each cell type within the printed
constructs, hAFSC cells were induced to differentiate into
osteogenic lineage followed by evaluation of calcium production
using Alizarin red staining Smooth muscle cell function was
assessed by measuring the resting membrane potentials and K.sup.+
currents using a patch clamp system (Axopatch 200B).
[0081] Results and Discussion. Fabrication of multi-cellular
structures. All three printed cell types were confirmed by their
corresponding cell identification methods, as shown in FIG. 1. The
GFP labeled MS1 cells exhibited green fluorescence and smooth
muscle cells emitted red under UV. The X-gal staining confirmed the
lacZ transfected hAFSC cells in blue under bright field microscopy.
All three cell types were present in an organized fashion within
the printed construct. A 3-D collagen "pie" with different color
dyes was shown in FIG. 1E, demonstrating the capability of the
inkjet printers to print different biomaterials as well as multiple
cell types.
[0082] Functional evaluation. Alizarin red staining showed the
production of calcium in the osteogenic differentiation culture of
hAFSC (FIG. 2a), which suggests that hAFSC in the collagen
constructs retain their capability to differentiate into specific
cell lineages under appropriate conditions. The whole cell patch
clamp recording showed the average resting membrane potential of
the printed BSMC (-58.5.+-.5.8 mV), which is similar to normal
non-printed smooth muscle cells (-54.7.+-.7.5 mV). There was no
significant difference on the K.sup.+ I-V relationship between the
printed cells and the normal controls (FIG. 2b). These findings
demonstrate that smooth muscle cells in the printed collagen
constructs maintained their normal basic electrophysiological
properties.
[0083] Conclusions. This example shows that viable
three-dimensional heterogeneous constructs with multiple cell types
can be generated by printing multiple cells and collagen gels
layer-by-layer. These distinct cells are able to survive and
proliferate within the 3-D constructs, and maintain normal basic
cellular properties and function in their spatially registered
regions. These findings demonstrate the possibility of building
complex tissues that require multiple cell types and ECM materials
by using the bio-printing technology.
EXAMPLE 2
In Vivo Generation of Tissues with Ink-Jet Printing
[0084] In this example we investigated whether the printed
multi-cell derived tissue constructs could maintain their
structural and spatial orientation in vivo. We examined whether
these tissues are able to survive and mature into functional
tissues when implanted in vivo.
[0085] Materials and Methods: Three-dimensional multi-cell
constructs with a "pie" configuration were fabricated by
simultaneously printing 3 different cell types [canine bladder
smooth muscle cells (SMC), bovine aortal endothelial cells (EC),
and human amniotic fluid-derived stem cells (AFSC)] into
collagen/alginate gel. The cells were labeled with 3 different
membrane bound tracers, which include [PKH67 (red), PKH26 (green),
and CMHC (blue)], respectively, prior to printing. Individual cells
were also printed separately for additional testing. The printed 3D
constructs were subcutaneously implanted into athymic mice.
AFSC-printed constructs were cultured in osteogenic medium for 1
week before implantation in order to induce differentiation into
bone tissue. The implanted constructs were monitored by MRI and
micro-CT scanner over time (up to 18 weeks). The retrieved
engineered tissues were analyzed with confocal microscopy and
immunohistochemical studies. To evaluate the function of the
engineered muscle, electrophysiological properties were performed
with voltage clamp experiments.
[0086] Results and Discussion.
[0087] Printed multi-cell implants. A complete 3D "pie" shaped
construct containing 3 different cell types (red dye tagged SMCs,
green dye tagged ECs, and blue dye tagged AFSCs) was successfully
fabricated by the inkjet method (FIG. 3). The tissue structure of
the 3D "pie" was maintained 2 weeks after implantation (FIG. 3b).
The membrane bound tracers confirmed that the printed cells
remained viable in their pre-determined locations (Green dye
stained ECs and red dye stained SMCs; (FIG. 3c, d).
[0088] In vivo functional evaluation. The voltage clamp recording
showed that the printed SMCs exhibited similar patterns in the mean
current voltage (I-V) relationships before and after implantation
(FIG. 4a), which suggests that the SMCs are able to maintain normal
basic electrophysiological characteristics in vivo. Vascularization
of the EC-printed implants was evaluated by MRI scanning 8 weeks
post-implantation. After the gadolinium (Gd) contrast agent was
injected intravenously into the animal, contrast enhancement was
visualized within the implants, which indicates the presence of
vascular network within the implanted tissues. FIG. 4b shows
intensified MRI signals, and the degree of contrast enhancements is
denoted in different colors in the implants. The formation of
vascularization was reconfirmed by the presence of blood vessels,
which were positively expressed with the endothelial cell-specific
marker: vWF (FIG. 4d). These data suggest that EC-printed implants
are able to form functional vasculature. Micro CT scanning showed
that bone-like hard tissues were formed within the AFSC-printed
constructs 18 weeks post-implantation (FIG. 4c)
Immuonohistochemical analysis showed that the differentiated AFSCS
within the implant expressed a typical bone cell marker,
osteocalcin (FIG. 3d). These data suggest that the printed stem
cells within the constructs retain their ability to differentiate
into specific cell lineages and enhance formation of relevant
tissues under specific conditions.
[0089] Conclusions. This example shows that multi-cellular
constructs, generated by the inkjet method, are able to maintain
their structural and spatial orientation in vivo. The printed cells
are able to retain their cellular characteristics and tissue
function. The inkjet printing technology may become a standard
method of engineering functional tissues for clinical
applications.
[0090] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
* * * * *